To elucidate the decisive structural factors relevant for dipeptide±carrier interaction, the affinity of short amide and imide derivatives for the intestinal H + /peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly-Sar transport in Caco-2 cells. Dipeptides with proline or alanine in the C-terminal position displayed affinity constants (K i ) of 0.15±1.2 mm and 0.08±9.5 mm, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N-terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa-Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24±70%) and the affinity constants was observed. After correcting the K i values for the incompetent cis isomers, the K i corr values of most dipeptides were in a small range of 0.1±0.16 mm. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa-Pro and Xaa-Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys-Pro, Arg-Pro and Pro-Pro indicate that additional molecular factors affect their binding at PEPT1. The K i values obtained for the corresponding Xaa-Ala dipeptides support this conclusion.Potential substrates or inhibitors of peptide transport were found among Xaa-piperidides and Xaa-thiazolidides. Dipeptides with N-terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro-Xaa dipeptides such as Pro-Leu, Pro-Tyr and Pro-Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C-terminal amino acid and the rigidity of the structure.

This study was initiated to determine whether the intestinal H؉ /peptide symporter PEPT1 differentiates between the peptide bond conformers of substrates. We synthesized a modified dipeptide where the peptide bond is replaced by the isosteric thioxo peptide bond. The Ala-Pro derivative Ala-[CS-N]-Pro exists as a mixture of cis and trans conformation in aqueous solution and is characterized by a low cis/trans isomerization rate. The compound was recognized by PEPT1 with high affinity. The K i value of Ala-[CS-N]-Pro for the inhibition of the uptake of radiolabeled glycylsarcosine in Caco-2 cells was 0.30 ؎ 0.02 mM, determined in solution with 96% trans conformation. In contrast, the K i value was 0.51 ؎ 0.02 mM when uptake media with 62% trans conformer were used. We conclude that only the trans conformer interacts with the transport system. From our data, a significant affinity of the cis conformer at PEPT1 cannot be derived. In a second approach, conformer-specific uptake of Ala-[CS-N]-Pro was studied by analyzing the intracellular content of Caco-2 cells following transport as well as the composition of the extracellular medium using capillary electrophoresis. The percentage of trans conformer that was 62% in the uptake medium increased to 92% inside the cells. This is the first direct evidence that an H ؉

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